2′-arabino-fluorooligonucleotide N3′-P5′ phosphoramidates: their synthesis and use

ABSTRACT

Oligonucleotides with a novel sugar-phosphate backbone containing at least one 2′-arabino-fluoronucleoside and an internucleoside 3′-NH—P(═O)(OR)—O-5′ linkage, where R is a positively charged counter ion or hydrogen, and methods of synthesizing and using the inventive oligonucleotides are provided. The inventive phosphoramidate 2′-arabino-fluorooligonucleotides have a high RNA binding affinity to complementary nucleic acids and are base and acid stable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/181,823 filed May 29, 2003now U.S. Pat. No. 7,321,029, which is anational stage application of International Application. No.PCT/US01/01918 filed on Jan. 19, 2001, which claims priority from U.S.Application No. 60/178,248, filed Jan. 21, 2000, all of which are herebyincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to 2′-arabino-fluorooligonucleotideN3′→P5′ phosphoramidates. More particularly, the invention is directedto 2′-arabino-fluoro monomer and oligonucleotide phosphoramidatecompositions, their use as diagnostic or therapeutic agents and methodsfor synthesizing phosphoramidate oligonucleotides containing said2′-arabino-fluorooligonucleotides.

BACKGROUND OF THE INVENTION

Nucleic acid polymer chemistry has played a crucial role in manydeveloping technologies in the pharmaceutical, diagnostic, andanalytical fields, and more particularly in the subfields of antisenseand anti-gene therapeutics, combinatorial chemistry, branched DNA signalamplification, and array-based DNA diagnostics and analysis (e.g.,Uhlmann and Peyman, Chemical Reviews, 90:543-584, 1990; Milligan et al.,J. Med. Chem. 36:1923-1937, 1993; DeMesmaeker et al., Current Opinion inStructural Biology, 5:343-355, 1995; Roush, Science, 276:1192-1193,1997; Thuong et al., Angew. Chem. Int. Ed. Engl., 32:666-690, 1993;Brenner et al., Proc. Natl. Acad. Sci., 89:5381-5383, 1992; Gold et al.,Ann. Rev. Biochem., 64.763-797, 1995; Gallop et al., J. Med. Chem.,37:1233-1258, 1994; Gordon et al., J. Med. Chem., 37:1385-1401, 1994;Gryaznov, International application No. PCT/US94/07557; Urdea et al.,U.S. Pat. No. 5,124,246; Southern et al., Genomics, 13:1008-1017, 1992;McGall et al., U.S. Pat. No. 5,412,087; Fodor et al., U.S. Pat. No.5,424,186; Pirrung et al., U.S. Pat. No. 5,405,783).

Much of this chemistry has been directed to improving the bindingstrength, specificity, and nuclease resistance of natural nucleic acidpolymers, such as DNA. Unfortunately, improvements in one property, suchas nuclease resistance, often involve trade-offs against otherproperties, such as binding strength. Examples of such trade-offsabound: peptide nucleic acids (PNAs) display good nuclease resistanceand binding strength, but have reduced cellular uptake in test cultures(e.g., Hanvey et al., Science, 258:1481-1485, 1992); phosphorothioatesdisplay good nuclease resistance and solubility, but are typicallysynthesized as P-chiral mixtures and display severalsequence-non-specific biological effects (e.g., Stein et al., Science,261:1004-1012, 1993); methylphosphonates display good nucleaseresistance and cellular uptake, but are also typically synthesized asP-chiral mixtures and have reduce duplex stability (e.g., DeMesmaeker etal. (cited above); and so on.

Recently, a new class of oligonucleotide analog has been developedhaving so-called N3′→P5′ phosphoramidate internucleoside linkages whichdisplay favorable binding properties, nuclease resistance, andsolubility (Gryaznov and Letsinger, Nucleic Acids Research,20:3403-3409, 1992; Chen et al., Nucleic Acids Research, 23:2661-2668,1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802, 1995; andGryaznov et al., J. Am. Chem. Soc., 116:3143-3144, 1994).Phosphoramidate compounds contain a 3′-amino group at each of the2′-deoxyfuranose nucleoside residues replacing a 3′-oxygen atom. Thesynthesis and properties of oligonucleotide N3′→P5′ phosphoramidates arealso described in Gryaznov et al., U.S. Pat. Nos. 5,591,607; 5,599,922;5,726,297; and Hirschbein et al., U.S. Pat. No. 5,824,793.

Oligonucleotides with various modifications of the internucleosidelinkages and 2′-position of the sugar rings have been described. Amongthese compounds are phosphodiester (PO), and phosphorothioate (PS)oligonucleotides containing 2′-fluoro substituents in ribo- or inarabino-configurations (Kawasaki et al., J. Med. Chem. 36:831-841, 1993;Ikeda et al., Nucl. Acids Res., 26:2217-2244, 1998; Kois et al.,Nucleosides Nucleotides, 12:1093-1109, 1993). Of these theoligo-2′-ribo-fluoronucleotides form the most stable complexes with DNAand RNA, whereas stability of duplexes formed by the isomericoligo-2′-arabino-fluoro nucleotides is significantly lower. The duplexstabilizing effects of 2′-ribo-fluoronucleotides was mainly attributedto their C3′-endo or N-type sugar puckering (Kawasaki et al., J. Med.Chem. 36:831-841, 1993). Unfortunately, phosphodiesteroligo-2′-ribo-fluoronucleotides are not resistant to hydrolysis bycellular nucleases, and require further modification of theinternucleoside linking groups for any in vivo applications of thesecompounds. Therefore, more stable oligonucleotide phosphorothioate(Kawasaki et al., J. Med. Chem. 36:831-841, 1993) and N3′→P5′phosphoramidates (Schultz et al., Nucl. Acids Res., 24:2966-2973, 1996),which resist enzymatic digestion were prepared. For the former compoundsintroduction of phosphorothioate linkages resulted in reduction of theduplex stability, whereas for the latter ones synergistic stabilizingeffects of both 2′-ribo-fluoro and 3′-amino groups were observed.Additionally, oligo-2′-ribo-fluoro-N3′→P5′ phosphoramidates were lessacid labile than their 2′-deoxy N3′→P5′ phosphoramidate counterparts(Schultz et al., Nucl. Acids Res., 24:2966-2973, 1996).

The oligonucleotide N3′→P5′ phosphoramidates form unusually stableduplexes with complementary DNA and especially RNA strands, as well asstable triplexes with DNA duplexes, and they are also resistant tonucleases (Chen et al., Nucleic Acids Research, 23:2661-2668, 1995;Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802 1995). Moreoveroligonucleotide N3′→P5′ phosphoramidates were found to be more potentantisense agents than phosphorothioate derivatives both in vitro and invivo (Skorski et al., Proc. Natl. Acad. Sci., 94:3966-3971, 1997). Atthe same time the phosphoramidates apparently have a low affinity to theintra- and extracellular proteins and increased acid liability relativeto the natural phosphodiester counterparts (Gryaznov et al., NucleicAcids Research, 24:1508-1514, 1996). These two features of theoligonucleotide phosphoramidates may potentially adversely effect theirpharmacological properties for some applications. In particular, theacid stability of an oligonucleotide is an important quality given thedesire to use oligonucleotide agents as oral therapeutics.

In order to circumvent the above described problems associated withpresently known oligonucleotide analogs, a new class of compounds wassought that embodies the best characteristics from both oligonucleotidephosphoramidates and 2′-ribo-fluoronucleotides. The present inventiondescribes the synthesis, properties and uses of oligonucleotideanalogues containing 2′-arabino-fluoronucleosides and internucleosideN3′→P5′ phosphoramidate linkages.

SUMMARY OF THE INVENTION

The compositions and methods of the present invention relate tonucleosides and to polynucleotides having contiguous nucleoside subunitsjoined by intersubunit linkages. In one aspect the present inventionrelates to 2′-arabino-fluoronucleoside and to polynucleotides comprisinga plurality of nucleoside subunits comprising at least one2′-arabino-fluoronucleoside linked to at least one additional nucleosidesubunit by a N3′→P5′ phosphoramidate inter-subunit linkage. Thepolynucleotides of the present invention preferably contain at least one2′-arabino-fluoronucleoside subunit joined by a N3′→P5′ phosphoramidateintersubunit linkage defined by the formula of 3′-[-NH—P(—O)(OR)—O-]-5′,wherein R is a positively charged counter ion, hydrogen, or lower alkyl.In a preferred embodiment of the invention, R is hydrogen. The inventivepolynucleotides can be composed such that all of the intersubunitlinkages are N3′→P5′ phosphoramidates. Alternatively, thepolynucleotides of the invention contain a second class of intersubunitlinkages such as phosphodiester, phosphotriester, methylphosphonate,P3′→N5′ phosphoramidate, N3′→P5′ thiophosphoramidate, andphosphorothioate linkages.

An exemplary N3′→P5′ 2′-arabino-fluoro phosphoramidate oligonucleotidehas the formula:

where B is a purine or pyrimidine or an analog thereof, n is an integerbetween 1 and 49, R is a positively charged counter ion, hydrogen, orlower alkyl, and R₁ is selected from the group consisting of hydroxyl,amino and hydrogen. The nucleoside subunits making up thepolynucleotides of the present invention can be selected to be in adefined sequence: such as, a sequence of bases complementary to asingle-strand nucleic acid target sequence or a sequence that will allowformation of a triplex structure between the polynucleotide and a targetduplex. The inventive oligonucleotides having 2′-arabino-fluorophosphoramidate subunits and N3′→P5′ phosphoramidate intersubunitlinkages, as described above, have superior resistance to acidhydrolysis, yet retain the same thermal stability as compared tooligonucleotides containing 2′-ribo-fluoronucleotides joined byphosphodiester linkages.

The present invention also includes a method of synthesizing anoligonucleotide containing 2′-arabino-fluoronucleosides andinternucleoside N3′→P5′ phosphoramidate linkages. In this method a first3′-amino protected nucleoside is attached to a solid phase support. Theprotected 3′ amino group is then deprotected to form a free 3′ aminogroup to which a second nucleoside is added. The free 3′ amino group ofthe first nucleoside is reacted with a 3′-protectedaminonucleoside-5′-(O-cyanoethyl-N,N-diisopropylamino)-2′-arabino-fluorophosphoramidite monomer to form an oligonucleotide internucleosideN3′→P5′ phosphoramidite linkage. The phoshoramidite linkage is thenoxidized to form a phosphoramidate internucleoside linkage.

The present invention also provides 2-arabino-fluoro monomers offormula:

where B is a purine or pyrimidine or an analog thereof; R₂ is H, loweralkyl, PO₃, or PN(R₄)₂OR₅ wherein R₄ is dialkyl, and R₅ is cyano-loweralkyl; and R₃ is hydrogen or substituted or unsubstituted trityl. In onerepresentative embodiment, R₂ is PN(R₄)₂OR₅ wherein R₄ is diisopropyl,R₅ is O-cyanoethyl and R₃ is monomethoxytrityl, as shown in the formulabelow:

In another embodiment the constituent B is exocyclic amino protected. Inother embodiments of the invention, when B is guanine the N2 amino groupof guanine is protected with an isobutyl group, when B is2,6-diaminopurine the exocylic amine groups are protected with aphenoxyancetyl group, and when B is cytosine the N4 amino group ofcytosine is protected with a benzoyl group.

In another embodiment of the invention, a method is provided forhybridizing a 2′-arabino-fluoro oligonucleotide N3′→P5′ phosphoramidateto a nucleic acid target. First a polynucleotide comprising a definedsequence of nucleoside subunits defined by the formula:

where B is a purine or pyrimidine or an analog thereof, n is an integerbetween 1 and 49 and R₁ is selected from the group consisting ofhydroxyl, amino and hydrogen. The 2′ arabino-fluoro N3′→P5′phosphoramidate polynucleotide is then contacted with the nucleic acidtarget to allow formation of a hybridization complex between thepolynucleotide and the nucleic acid target.

The present invention also includes pharmaceutical compositions and kitsfor the isolation of a target RNA that include a polynucleotide havingat least one 2′-arabino-fluoronucleoside and phosphoramidate N3′→P5′linkage, as described above. The inventive oligonucleotides areparticularly useful in oral therapeutic applications based onhybridization, such as, antigene and antisense applications, includingthe inhibition of telomerase enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic outline of the step-by-step synthesis of2-arabino-fluoronucleoside monomers, B₁ corresponds to thymine oruracil, B₂ corresponds to thymine, uracil or N⁴-benzoyl-cytosine and R1is hydrogen or methyl;

FIG. 2 shows the overall synthetic scheme used to prepare the protectedpurine 2′-arabino-fluoro phosphoramidite monomers of the presentinvention. B represents a base selected from the group consisting ofadenine (A), guanine (G), and 2,6-diaminopurine (D), uracil (U),cytosine (C) and thymidine (T). Lower case letters a-d associated withcompound numbers represent the bases adenine (a), guanine (g),2,6-diaminopurine (d), uracil (u) and thymine (t). Tol is touoyl, MMTNHis (monomethoxytrityl)amino, iPr₂N is diisopropylamino, and CEO isβ-cyanoethyl, R is anisoyl when the base is G or D, and toluoyl when thebase is A, T, or U. In addition, when B is adenine the N6 amino group ofadenine is protected with a benzoyl group, when B is 2,6-diaminopurinethe exocylic amine groups are protected with a phenoxyacetyl group, orwhen B is guanine the N² amino group of guanine is protected with anisobutyl group; and

FIG. 3 shows the internucleoside linkage structure of an oligonucleotidecontaining 2′-arabino-fluoronucleosides joined an internucleosideN3′→P5′ phosphoramidate linkage, where R is a positively charged counterion or hydrogen;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

An “alkyl group” refers to an alkyl or substituted alkyl group having 1to 20 carbon atoms, such as methyl, ethyl, propyl, and the like. Loweralkyl typically refers to C₁ to C₅. Intermediate alkyl typically refersto C₆ to C₁₀.

An “aryl group” refers to an aromatic ring group having 5-20 carbonatoms, such as phenyl, naphthyl, anthryl, or substituted aryl groups,such as alkyl- or aryl-substitutions like tolyl, ethylphenyl,biphenylyl, etc. Also included are heterocyclic aromatic ring groupshaving one or more nitrogen, oxygen, or sulfur atoms in the ring.

A “positively charged counter ion” refers to any ion capable of formingan ion pair with oxygen, such a Na⁺, K⁺, Ca⁺, Mg²⁺, Mn²⁺ and the like.

“Oligonucleotides” typically refer to nucleoside subunit polymers havingbetween about 2 and about 50 contiguous subunits. The nucleosidesubunits can be joined by a variety of intersubunit linkages, including,but not limited to, those shown in FIGS. 1A to 1C. Further,“oligonucleotides” includes modifications, known to one skilled in theart, to the sugar backbone (e.g., ribose or deoxyribose subunits), thesugar (e.g., 2′ substitutions), the base, and the 3′ and 5′ termini. Theterm “polynucleotide”, as used herein, has the same meaning as“oligonucleotide” is used interchangeably with “polynucleotide”.

Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGUCCTG,” it will be understood that the nucleotides are in5′→3′ order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotesthymidine, and “U” denotes deoxyuridine, unless otherwise noted.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g., as described in Kornberg andBaker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified base moieties and/or modified sugar moieties, e.g.,described generally by Scheit, Nucleotide Analogs (John Wiley, New York,1980). Such analogs include synthetic nucleosides designed to enhancebinding properties, e.g., stability, specificity, or the like, such asdisclosed by Uhlmann and Peyman (Chemical Reviews, 90:543-584, 1990).

A “base” is defined herein to include (i) typical DNA and RNA bases(uracil, thymine, adenine, guanine, and cytosine), and (ii) modifiedbases or base analogs (e.g., 5-methyl-cytosine, 5-bromouracil, orinosine). A base analog is a chemical whose molecular structure mimicsthat of a typical DNA or RNA base.

As used herein, “pyrimidine” means the pyrimidines occurring in naturalnucleosides, including cytosine, thymine, and uracil, and common analogsthereof, such as those containing oxy, methyl, propynyl, methoxy,hydroxyl, amino, thio, halo, and like substituents. The term as usedherein further includes pyrimidines with common protection groupsattached, such as N₄-benzoylcytosine. Further common pyrimidineprotection groups are disclosed by Beaucage and Iyer (Tetrahedron48:223-2311, 1992).

As used herein, “purine” means the purines occurring in naturalnucleosides, including adenine, guanine, and hypoxanthine, and commonanalogs thereof, such as those containing oxy, methyl, propynyl,methoxy, hydroxyl, amino, thio, halo, and like substituents. The term asused herein further includes purines with common protection groupsattached, such as N₂-benzoylguanine, N₂-isobutyrylguanine,N₆-benzoyladenine, and the like. Further common purine protection groupsare disclosed by Beaucage and Iyer (cited above).

As used herein, the term “protected” as a component of a chemical namerefers to art-recognized protection groups for a particular moiety of acompound, e.g., “5′-protected-hydroxyl” in reference to a nucleosideincludes triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl (i.e.,monomethoxytrityl or MMT), di-p-anisylphenylmethyl (i.e.,dimethoxytrityl or DMT), and the like. Art-recognized protection groupsare described in the following references: Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); Amamath andBroom, Chemical Reviews, 77:183-217, 1977; Pon et al., Biotechniques,6:768-775, 1988; Ohtsuka et al., Nucleic Acids Research, 10:6553-6570,1982; Eckstein, editor, Oligonucleotides and Analogues: A PracticalApproach (IRL Press, Oxford, 1991), Greene and Wuts, Protective Groupsin Organic Synthesis, Second Edition (John Wiley & Sons, New York,1991), Narang, editor, Synthesis and Applications of DNA and RNA(Academic Press, New York, 1987), Beaucage and Iyer (cited above), andlike references.

As used herein, “stringency” refers to the hybridization conditionsunder which an oligonucleotide binds to a nucleic acids to which it hassequence homology, i.e. a “target nucleic acid.” It is understood thatan oligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of utility, and there is a sufficient degree of complementarity toavoid nonspecific binding of oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment or, in the case of in vitro assays, under conditions in whichthe assays are conducted.

The term “hybridization stringency” is well known in the art and relatesto the approximate buffer, salt and temperature conditions under whichan oligonucleotide hybridizes specifically to its target nucleic acid.Generally, the following conditions are used to define hybridizationstringency: “high stringency” denotes the use of a hybridization or washsolution comprising 10 mM phosphate buffer, pH 7.0, at a range of about45-55° C. The term “moderate stringency” means use of 10 mM phosphatebuffer, pH 7.0, with a salt concentration of about 0.1 to 0.5 M NaCl, ata temperature of between about 30 to 45° C. The term “low stringency”means use of about 10 mM phosphate buffer at about pH 7.0, 1.0 M NaCl atroom temperature. Low stringency buffers may also include 10 mM MgCl₂.It is well understood in the art of nucleic acid hybridization that manyfactors, such as temperature, salt and inclusion of other componentssuch as formamide, affect the stringency of hybridization.

The compounds of the present invention may be used to inhibit or reduceenzyme activity, such as reducing the activity of the telomerase enzymeand/or reducing the proliferation of cells having telomerase activity.In these contexts, inhibition or reduction of the enzyme activity orcell proliferation refer to a lower level of the measured activityrelative to a control experiment in which the enzyme or cells are nottreated with the test compound. In particular embodiments, theinhibition or reduction in the measured activity is at least a 10%reduction or inhibition. One of skill in the art will appreciate thatreduction or inhibition of the measured activity of at least 20%, 50%,75%, 90% or 100% may be preferred for particular applications.

The present invention is directed generally to2′-arabino-fluorooligonucleosides and to oligonucleotides containing atleast one 2′-arabino-fluoronucleoside joined by an internucleosideN3′→P5′ phosphoramidate linkage, methods of synthesizing such analogpolynucleotides, and methods of using the inventive oligonucleotides astherapeutic compounds and in diagnostics. More particularly, the2′-arabino-fluorooligonucleotides of the present invention have theformula:

wherein B is a purine or pyrimidine or an analog thereof such as uracil,thymine, adenine, guanine, cytosine, 5-methylcytosine, 5-bromouracil andinosine,

R is a positively charged counter ion or hydrogen,

R₁ is selected from the group consisting of hydroxyl, amino andhydrogen, and

n is an integer between 1 and 49.

The nucleoside subunits making up the polynucleotides nucleotides of thepresent invention can be selected to be in a defined sequence: such as,a sequence of bases complementary to a single-strand nucleic acid targetsequence or a sequence that will allow formation of a triplex structurebetween the polynucleotide and a target duplex.

The inventive oligonucleotides can be used to hybridize to targetnucleic acid sequences such as RNA and DNA. When desirable, theoligonucleotides of the present invention can be labeled with a reportergroup, such as radioactive labels, biotin labels, fluorescent labels andthe like, to facilitate the detection of the polynucleotide itself andits presence in, for example, hybridization complexes.

In another aspect of the invention, a kit for isolating a target nucleicacid from a sample is provided. The kit contains an oligonucleotidehaving a defined sequence of nucleoside subunits joined by a least oneintersubunit linkage defined by the formula:

where B is a purine or pyrimidine or an analog thereof, n is an integerbetween 1 and 49, R is a positively charged counter ion or hydrogen, andR₁ is selected from the group consisting of hydroxyl, amino andhydrogen, and wherein the oligonucleotide hybridizes to the targetnucleic acid.

In other aspects, the invention is directed to a solid phase method ofsynthesizing oligonucleotide containing 2′-arabino-fluoronucleosides andinternucleoside N3′→P5′ phosphoramidate linkages using a modification ofthe phosphoramidite transfer methodology of Nelson et al. (J. OrganicChemistry 62:7278-7287, 1997). The synthetic strategy employed3′—NH-trityl-protected 3′-aminonucleoside5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidites (Nelson et al.,cited above) that were purchased from Cruachem and JBL Scientific, Inc.(Aston, Pa. and San Luis Obispo, Calif., respectively). Every syntheticcycle was conducted using the following chemical steps: 1)detritylation, 2) coupling; 3) capping, and 4) oxidizing.

Chimeric 2′-arabino-fluoro N3′→P5′-thiophosphoramidate oligonucleotidescan be made by substitution of a sulfurization reaction in place of theoxidation reaction at synthetic step 4 above, which results in formationof a thio-phosphoramidate mixed oligonucleotide (see Pongracz, et al.,Tetrahedron, 40:7661-7664, 1999). In addition, chimeric oligonucleotidescan be made comprising 2′-ribo-fluoro (see Schultz, et al., Nucl. AcidsRes., 24:2966-2973, 1996) and 2′-arabino-fluoro phosphoramidates, andthio-N3′→P5′-phosphoramidates with 2′-arabino-fluoronucleosides.Similarly, phosphodiester-2′-arabino-fluoro phosphoramidates can be madeby using 5′-phosphoramidite-3′-O-DMTr-protected nucleotides as monomericbuilding blocks. These synthetic approaches are known in the art (seePongracz, et al., 1999 and Schultz, et al., 1996.).

In another embodiment of the present invention, the acid stability ofoligonucleotides is increased by incorporating2′-arabino-fluoronucleosides subunits lined by N3′→P5′thiophosphoramidate intersubunit linkages into an oligonucleotide. Thehybridization properties of the phosphoramidate2′-arabino-fluorooligonucleotides were evaluated relative tocomplementary DNA or RNA strands having phosphodiester or2′-arabino-ribose phosphoramidate intersubunit linkages. The thermalstability data for duplexes generated from 2′-arabino-ribosephosphoramidate, phosphoramidate, phosphodiester and the inventive2′-arabino-fluoro phosphoramidate oligonucleotides, are summarized inExample 4, Table 1.

Applications of Oligonucleotides Containing 2-Arabino-Fluoronucleosidesand Internucleoside 3′-NHP(O)(O⁻)O-5′ Phosphoramidate Linkages

2′-Arabino-fluorooligonucleotide SEQ ID NO:8 phosphoramidate wassynthesized (see Table 1). This compound was surprisingly base and acidstable and formed a stable complex with a complementary RNA and DNAtarget The N3′→P5′ phosphoramidate 2′-arabino-fluoro-polynucleotides ofthe present invention are useful for anti-sense and anti-genediagnostic/therapeutic applications. In a preferred embodiment of thepresent invention, the oligonucleotides are oligodeoxyribonucleotides.

A. Telomerase Inhibition Applications

Recently, an understanding of the mechanisms by which normal cells reachthe state of senescence, i.e., the loss of proliferative capacity thatcells normally undergo in the cellular aging process, has begun toemerge. The DNA at the ends, or telomeres, of the chromosomes ofeukaryotes usually consists of tandemly repeated simple sequences.Scientists have long known that telomeres have an important biologicalrole in maintaining chromosome structure and function. More recently,scientists have speculated that the cumulative loss of telomeric DNAover repeated cell divisions may act as a trigger of cellular senescenceand aging, and that the regulation of telomerase, an enzyme involved inthe maintenance of telomere length, may have important biologicalimplications. See Harley, Mutation Research, 256:271-282, 1991.Experiments by Bodnar et al. have confirmed the importance of telomeresand telomerase in controlling the replicative lifespan of culturednormal human cells. See Bodnar et al., Science 279:349-352, 1998.

Telomerase is a ribonucleoprotein enzyme that synthesizes one strand ofthe telomeric DNA using as a template a sequence contained within theRNA component of the enzyme. See Blackburn, Annu. Rev. Biochem.,61:113-129, 1992. The RNA component of human telomerase has beensequenced and is 453 nucleotides in length containing a series of11-base sequence repeats that is complementary to the telomere repeat.Human telomerase activity has been inhibited by a variety ofoligonucleotides complementary to the RNA component of telomerase. SeeNorton et al., Nature Biotechnology, 14:615, 1996; Pitts et al., Proc.Natl. Acad. Sci., 95:11549-11554, 1998; and Glukhov et al., Bioch.Biophys. Res. Commun., 248:368-371, 1999. 2-Arabino-fluorophosphoramidate oligonucleotides of the present invention arecomplementary to 10 to 50 nucleotides of telomerase RNA. Preferably, theinventive telomerase inhibitor 2-arabino-fluoro phosphoramidateoligonucleotides have a 10 to 20 consecutive base sequence that arecomplementary to telomerase RNA.

Methods for detecting telomerase activity, as well as for identifyingcompounds that regulate or affect telomerase activity, together withmethods for therapy and diagnosis of cellular senescence andimmortalization by controlling telomere length and telomerase activity,have also been described. See, Feng et al., Science, 269:1236-1241,1995; Kim et al., Science, 266:2011-2014, 1994; PCT patent publicationNo. 93/23572, published Nov. 25, 1993; and U.S. Pat. Nos. 5,656,638,5,760,062, 5,767,278, 5,770,613 and 5,863,936.

The identification of compounds that inhibit telomerase activityprovides important benefits to efforts at treating human disease.Compounds that inhibit telomerase activity can be used to treattelomerase-mediated disorders, such as cancer, since cancer cellsexpress telomerase activity and normal human somatic cells do notpossess telomerase activity at biologically relevant levels (i.e., atlevels sufficient to maintain telomere length over many cell divisions).Unfortunately, few such compounds, especially compounds with highpotency or activity and compounds that are orally bioavailable, havebeen identified and characterized. Hence, there remains a need forcompounds that act as telomerase inhibitors that have relatively highpotency or activity and that are orally bioavailable, and forcompositions and methods for treating cancer and other diseases in whichtelomerase activity is present abnormally.

The new phosphoramidate 2′-arabino-fluorooligonucleotide compounds ofthe present invention are acid and base stable, and therefore, have manyvaluable uses as inhibitors of deleterious telomerase activity, such as,for example, in the treatment of cancer in humans. Pharmaceuticalcompositions of phosphoramidate 2′-arabino-fluorooligonucleotide can beemployed in treatment regimens in which cancer cells are killed, invivo, or can be used to kill cancer cells ex vivo. Thus, this inventionprovides therapeutic compounds and compositions for treating cancer, andmethods for treating cancer in mammals (e.g., cows, horses, sheep,steer, pigs and animals of veterinary interest such as cats and dogs).In addition, the phosphoramidate 2′-arabino-fluorooligonucleotides ofthe present invention may also be used to treat othertelomerase-mediated conditions or diseases, such as, for example, otherhyperproliferative or autoimmune disorders such as psoriasis, rheumatoidarthritis, immune system disorders requiring immune system suppression,immune system reactions to poison ivy or poison oak, and the like.

As noted above, the immortalization of cells involves inter alia theactivation of telomerase. More specifically, the connection betweentelomerase activity and the ability of many tumor cell lines, includingskin, connective tissue, adipose, breast, lung, stomach, pancreas,ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervoussystem (CNS), retina and blood tumor cell lines, to remain immortal hasbeen demonstrated by analysis of telomerase activity (Kim et al.,above). This analysis, supplemented by data that indicates that theshortening of telomere length can provide the signal for replicativesenescence in normal cells, see PCT Application No. 93/23572,demonstrates that inhibition of telomerase activity can be an effectiveanti-cancer therapy. Thus, telomerase activity can prevent the onset ofotherwise normal replicative senescence by preventing the normalreduction of telomere length and the concurrent cessation of cellreplication that occurs in normal somatic cells after many celldivisions. In cancer cells, where the malignant phenotype is due to lossof cell cycle or growth controls or other genetic damage, an absence oftelomerase activity permits the loss of telomeric DNA during celldivision, resulting in chromosomal rearrangements and aberrations thatlead ultimately to cell death. However, in cancer cells havingtelomerase activity, telomeric DNA is not lost during cell division,thereby allowing the cancer cells to become immortal, leading to aterminal prognosis for the patient. Agents capable of inhibitingtelomerase activity in tumor cells offer therapeutic benefits withrespect to a wide variety of cancers and other conditions (e.g., fungalinfections) in which immortalized cells having telomerase activity are afactor in disease progression or in which inhibition of telomeraseactivity is desired for treatment purposes. The telomerase inhibitors ofthe invention can also be used to inhibit telomerase activity in germline cells, which may be useful for contraceptive purposes.

In addition, it will be appreciated that therapeutic benefits fortreatment of cancer can be realized by combining a telomerase inhibitorof the invention with other anti-cancer agents, including otherinhibitors of telomerase such as described in U.S. Pat. Nos. 5,656,638,5,760,062, 5,767,278, 5,770,613 and 5,863,936. The choice of suchcombinations will depend on various factors including, but not limitedto, the type of disease, the age and general health of the patient, theaggressiveness of disease progression, the TRF length and telomeraseactivity of the diseased cells to be treated and the ability of thepatient to tolerate the agents that comprise the combination. Forexample, in cases where tumor progression has reached an advanced state,it may be advisable to combine a telomerase inhibiting compound of theinvention with other agents and therapeutic regimens that are effectiveat reducing tumor size (e.g., radiation, surgery, chemotherapy and/orhormonal treatments). In addition, in some cases it may be advisable tocombine a telomerase inhibiting agent of the invention with one or moreagents that treat the side effects of a disease, e.g., an analgesic, oragents effective to stimulate the patient's own immune response (e.g.,colony stimulating factor).

The compounds of the present invention demonstrate inhibitory activityagainst telomerase activity in vivo, as can be demonstrated as describedbelow. The in vitro activities of the compounds of the invention canalso be demonstrated using the methods described herein. As used herein,the term “in vitro” refers to tests performed using living cells intissue culture. Such procedures are also known as “ex vivo”.

One method used to identify phosphoramidate 2′-arabino-fluoropolynucleotides of the invention that inhibit telomerase activityinvolves placing cells, tissues, or preferably a cellular extract orother preparation containing telomerase in contact with several knownconcentrations of a phosphoramidate 2′-arabino-fluorooligonucleotidethat is complementary to the RNA component of telomerase in a buffercompatible with telomerase activity. The level of telomerase activityfor each concentration of the phosphoramidate 2′-arabino-fluoropolynucleotide is measured and the IC₅₀ (the concentration of thepolynucleotide at which the observed activity for a sample preparationis observed to fall one-half of its original or a control value) for thepolynucleotide is determined using standard techniques. Other methodsfor determining the inhibitory concentration of a compound of theinvention against telomerase can be employed as will be apparent tothose of skill in the art based on the disclosure herein.

With respect to the treatment of malignant diseases usingphosphoramidate 2′-arabino-fluoro polynucleotides that are complementaryto the RNA component of telomerase are expected to induce crisis intelomerase-positive cell lines. Treatment of telomerase-positive celllines, such as HEK-293, HeLa and HME50-5E human breast epithelial cellsthat were spontaneously immortalized, with a phosphoramidate′-arabino-fluorooligonucleotide that is complementary to the RNAsequence component of telomerase is also expected to induce a reductionof telomere length in the treated cells.

Phosphoramidate 2′-arabino-fluorooligonucleotides of the invention arealso expected to induce telomere reduction during cell division in humantumor cell lines, such as the ovarian tumor cell lines OVCAR-5 andSK-OV-3. Importantly, however, in normal human cells used as a control,such as BJ cells of fibroblast origin, the observed reduction intelomere length is expected to be no different from cells treated with acontrol substance, e.g., a thiophosphoramidate oligonucleotide that hasat least one single base mismatch with the complementary telomerase RNAtarget. The phosphoramidate 2′-arabino-fluorooligonucleotides of theinvention also are expected to demonstrate no significant cytotoxiceffects at concentrations below about 20 μM in the normal cells.

In addition, the specificity of the phosphoramidate2′-arabino-fluorooligonucleotides of the present invention fortelomerase RNA can be determined by performing hybridization tests withand comparing their activity (IC₅₀) with respect to telomerase and toother enzymes known to have essential RNA components. Compounds havinglower IC₅₀ values for telomerase as compared to the IC₅₀ values towardthe other enzymes being screened are said to possess specificity fortelomerase.

In vivo testing can also be performed using a mouse xenograft model, forexample, in which OVCAR-5 tumor cells are grafted onto nude mice, inwhich mice treated with a phosphoramidate2′-arabino-fluorooligonucleotide of the invention are expected to havetumor masses that, on average, may increase for a period following theinitial dosing, but will begin to shrink in mass with continuingtreatment. In contrast, mice treated with a control (e.g., aphosphoramidate 2′-arabino-fluorooligonucleotide that has at least onesingle base mismatch with the complementary telomerase RNA target) areexpected to have tumor masses that continue to increase.

From the foregoing those skilled in the art will appreciate that thepresent invention also provides methods for selecting treatment regimensinvolving administration of a phosphoramidate2′-arabino-fluorooligonucleotide of the invention. For such purposes, itmay be helpful to perform a terminal restriction fragment (TRF) analysisin which DNA from tumor cells is analyzed by digestion with restrictionenzymes specific for sequences other than the telomeric (T₂ AG₃)_(N)sequence. Following digestion of the DNA, gel electrophoresis isperformed to separate the restriction fragments according to size. Theseparated fragments are then probed with nucleic acid probes specificfor telomeric sequences to determine the lengths of the terminalfragments containing the telomere DNA of the cells in the sample. Bymeasuring the length of telomeric DNA, one can estimate how long atelomerase inhibitor should be administered and whether other methods oftherapy (e.g., surgery, chemotherapy and/or radiation) should also beemployed. In addition, during treatment, one can test cells to determinewhether a decrease in telomere length over progressive cell divisions isoccurring to demonstrate treatment efficacy.

Thus, in one aspect, the present invention provides compounds that canserve in the war against cancer as important weapons against many typesof malignancies. In particular, the phosphoramidate2′-arabino-fluoro-polynucleotides of the present invention can provide ahighly general method of treating many, if not most, malignancies, asdemonstrated by the highly varied human tumor cell lines and tumorshaving telomerase activity. More importantly, the phosphoramidate2′-arabino-fluorooligonucleotides of the present invention can beeffective in providing treatments that discriminate between malignantand normal cells to a high degree, avoiding many of the deleteriousside-effects present with most current chemotherapeutic regimes whichrely on agents that kill dividing cells indiscriminately.

B. Antisense Applications

Antisense therapy involves the administration of exogenousoligonucleotides that bind to a target nucleic acid, typically an RNAmolecule, located within cells. The term antisense is so given becausethe oligonucleotides are typically complementary to mRNA molecules(“sense strands”) which encode a cellular product.

The phosphoramidate 2′-arabino-fluorooligonucleotides described hereinare useful for antisense inhibition of gene expression (Matsukura etal., Proc. Natl. Acad. Sci., 86:4244-4248, 1989; Agrawal et al., Proc.Natl. Acad. Sci., 86:7790-7794, 1989; Zamecnik et al., Proc. Natl. Acad.Sci., 83:4143-4146, 1986; Rittner and Sczakiel, Nucleic Acids Research,19:1421-1426, 1991; Stein and Cheng, Science, 261:1004-1012, 1993).N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides havetherapeutic applications for a large number of medically significanttargets, including, but not limited to inhibition of cancer cellproliferation and interference with infectious viruses. The N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotides are useful for bothveterinary and human applications. The high acid stability of theinventive oligonucleotides and their ability to act effectively asantisense molecules at low concentrations (see below) make theseoligonucleotides highly desirable as therapeutic antisense agents.

Anti-sense agents typically need to continuously bind all target RNAmolecules so as to inactivate them or alternatively provide a substratefor endogenous ribonuclease H (RNase H) activity. Sensitivity ofRNA/oligonucleotide complexes, generated by the methods of the presentinvention, to RNase H digestion can be evaluated by standard methods(Donia et al., J. Biol. Chem., 268:14514-14522, 1993; Kawasaki et al.,J. Medicinal Chem., 36:831-841, 1993).

The methods of the present invention provide several advantages over themore conventional antisense agents. First, phosphoramidate2′-arabino-fluorooligonucleotides bind more strongly to RNA targets ascorresponding phosphodiester, oligonucleotides. Second, thephosphoramidate 2′-arabino-fluorooligonucleotides are more resistant todegradation by cellular nucleases and by acid or base conditions.

Further, when an RNA is coded by a mostly purine strand of a duplextarget sequence, 2′-arabino-fluoro phosphoramidate analogoligonucleotides targeted to the duplex also have potential forinactivating the DNA—i.e., the ability to inactivate a pathogen in bothsingle-stranded and double-stranded forms (see discussion of anti-genetherapies below). Lastly, the oligonucleotides of the present inventionform more stable triple stranded structures when annealed to doublestranded DNA targets than do natural phosphodiester linkedoligonucleotides.

Sequence-specific phosphoramidate 2′-arabino-fluorooligonucleotidemolecules are potentially powerful therapeutics for essentially anydisease or condition that in some way involves RNA. Exemplary modes bywhich such sequences can be targeted for therapeutic applicationsinclude:

-   a) targeting RNA sequences expressing products involved in the    propagation and/or maintenance infectious agents, such as, bacteria,    viruses, yeast and other fungi, for example, a specific mRNA encoded    by an infectious agent;-   b) formation of a duplex molecule that results in inducing the    cleavage of the RNA (e.g., RNase H cleavage of RNA/DNA hybrid duplex    molecules). This is an important property of the inventive    2′-arabino-fluoro phosphoramidate oligonucleotides because, in    general, phosphoramidates are not good substrates for RNase H;-   c) blocking the interaction of a protein with an RNA sequence (e.g.,    the interaction of TAT and TAR, see below); and-   d) targeting sequences causing inappropriate expression or    proliferation of cellular genes: for example, genes associated with    cell cycle regulation; inflammatory processes; smooth muscle cell    (SMC) proliferation, migration and matrix formation (Liu et al.,    Circulation, 79:1374-1387, 1989); certain genetic disorders; and    cancers (protooncogenes).

In one embodiment, translation or RNA processing of inappropriatelyexpressed cellular genes is blocked. Exemplary potential targetsequences are protooncogenes, for example, including but not limited tothe following: c-myc, c-myb, c-fos, c-kit, ras, and BCR/ABL (e.g.,Wickstrom, Editor, Prospects for Antisense Nucleic Acid Therapy ofCancer and AIDS, Wiley-Liss, New York, N.Y., 1991; Zalewski et al.,Circulation Res., 88:1190-1195, 1993; Calabretta et al., Seminars inCancer Biol., 3:391-398, 1992; Calabretta et al., Cancer Treatment Rev.19:169-179, 1993), oncogenes/tumor suppresser genes (e.g., p53, Bayeveret al. Antisense Research and Development, 3:383-390, 1993),transcription factors (e.g., NF.kappa.B, Cogswell et al., J. Immunol.,150:2794-2804, 1993) and viral genes (e.g., papillomaviruses, Cowsert etal. Antimicrob. Agents and Chemo., 37:171-177, 1993; herpes simplexvirus, Kulka et al. Antiviral Res., 20:115-130, 1993). To furtherillustrate, two RNA regions of the HIV-1 protein that can be targeted bythe methods of the present invention are the REV-protein responseelement (RRE) and the TAT-protein transactivation response element(TAR). REV activity requires the presence of the REV response element(RRE), located in the HIV envelope gene (Malim et al., Nature,338:254-257, 1989; Malim et al., Cell, 58:205-214, 1989).

The RRE has been mapped to a 234-nucleotide region thought to form fourstem-loop structures and one branched stem-loop structure (Malim et al.,Nature, 338:254-257, 1989). Data obtained from footprinting studies(Holland et al., J. Virol., 64:5966-5975, 1990; Kjems et al., Proc.Natl. Acad. Sci., 88:683-687, 1991) suggest that REV binds to six basepairs in one stem structure and to three nucleotides in an adjacentstem-loop structure of the RRE. A minimum REV binding region of about 40nucleotides in stem-loop II has been identified by Cook et al. (NucleicAcids Research, 19:1577-1583). This binding region can be target forgeneration of RNA/DNA duplexes (e.g., Li et al., J. Virol.,67:6882-6888, 1993) using one or more phosphoramidate2′-arabino-fluorooligonucleotides, according to the methods of thepresent invention.

The HIV-1 TAT is essential for viral replication and is a potenttransactivator of long terminal repeat (LTR)-directed viral geneexpression (Dayton et al., Cell, 44:941-947, 1986; Fisher et al.,Nature, 320:367-371, 1986). Transactivation induced by TAT proteinrequires the presence of the TAR element (see U.S. Pat. No. 5,837,835)which is located in the untranslated 5′ end of the viral mRNA element.

The TAR element is capable of forming a stable stem-loop structure(Muesing et al., Cell, 48:691-701, 1987). The integrity of the stem anda 3 nucleotide (nt) bulge on the stem of TAR has been demonstrated to beessential for specific and high-affinity binding of the TAT protein tothe TAR element (Roy et al., Genes Dev., 4:1365-1373, 1990; Cordingleyet al., Proc. Natl. Acad. Sci., 87:8985-8989, 1990; Dingwall et al.,Proc. Natl. Acad. Sci., 86:6925-6929, 1989; Weeks et al., Science,249:1281-1285, 1990). This region can be targeted for anti-sense therapyfollowing the method of the present invention.

In addition to targeting the RNA binding sites of the REV, RRE and TATproteins, the RNA coding sequences for the REV and TAT proteinsthemselves can be targeted in order to block expression of the proteins.

Initial screening of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides, directed to bind potential antisensetarget sites, typically includes testing for the thermal stability ofresultant RNA/DNA duplexes. When a phosphoramidate2′-arabino-fluorooligonucleotide is identified that binds a selected RNAtarget sequence, the oligonucleotide is further tested for inhibition ofRNA function in vitro. Cell culture assays systems are used for such invitro analysis (e.g., herpes simplex virus, Kulka et al., AntiviralRes., 20:115-130, 1993; HIV-1, Li et al., J. Virol., 67:6882-6888, 1993;Vickers et al., Nucleic Acids Research, 19:3359-3368, 1991; coronarysmooth muscle cell proliferation in restenosis, Zalewski et al., NucleicAcids Research, 15:1699-1715, 1987; IL-2R, Grigoriev et al., Proc. Natl.Acad. Sci., 90:3501-3505, 1993; c-myb, Baer et al., Blood, 79:1319-1326,1992; c-fos, Cutry et al., J. Biol. Chem., 264:19700-19705, 1989;BCR/ABL, Szczylik et al., Science, 253:562-565, 1991).

C. Anti-Gene Applications

Inhibition of gene expression via triplex formation has been previouslydemonstrated (Cooney et al., Science, 241:456-459, 1989; Orson et al.,Nucleic Acids Research, 19:3435-3441, 1991; Postel et al., Proc. Natl.Acad. Sci., 88:8227-8231, 1991). The increased stability of triplexstructures formed when employing third strand phosphoramidate2′-arabino-fluorooligonucleotides provides a stronger tool for antigeneapplications, including veterinary and human therapeutic applications.

A target region of choice is selected based on known sequences usingstandard rules for triplex formation (Helene and Toulme, Biochem.Biophys. Acta, 1049:99-125, 1990). Typically, the phosphoramidate2′-arabino-fluorooligonucleotide sequence is targeted againstdouble-stranded genetic sequences in which one strand containspredominantly purines and the other strand contains predominantlypyrimidines.

Phosphoramidate 2′-arabino-fluorooligonucleotides of the presentinvention are tested for triplex formation against a selected duplextarget sequences using band shift assays (see for example, U.S. Pat. No.5,726,297, Example 4). Typically, high percentage polyacrylamide gelsare used for band-shift analysis and the levels of denaturing conditions(Ausubel et al., Current Protocols in Molecular Biology, Hohn Wiley andSons, Inc. Media Pa.; Sauer et al. eds., Methods in EnzymologyProtein/DNA Interactions, Academic Press, 1991; Sambrook et al. inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Vol.2, 1989) are adjusted to reduce any non-specific background binding.

The duplex target is labeled (for example, using a radioactivenucleotide) and mixed with a third strand oligonucleotide, being testedfor its ability to form triplex structures with the target duplex. Ashift of the mobility of the labeled duplex oligonucleotide indicatesthe ability of the oligonucleotide to form triplex structures.

Triplex formation is indicated in the band shift assay by a decreasedmobility in the gel of the labeled triplex structure relative to thelabeled duplex structure.

Numerous potential target sites can be evaluated by this methodincluding target sites selected from a full range of DNA sequences thatvary in length as well as complexity. Sequence-specific phosphoramidate2′-arabino-fluorooligonucleotide molecules are potentially powerfultherapeutics for essentially any disease or condition that in some wayinvolves DNA. Exemplary target sequences for such therapeutics include:a) DNA sequences involved in the propagation and/or maintenanceinfectious agents, such as, bacterial, viruses, yeast and other fungi,for example, disrupting the metabolism of an infectious agent; and b)sequences causing inappropriate expression or proliferation of cellulargenes, such as oncogenes, for example, blocking or reducing thetranscription of inappropriately expressed cellular genes (such as genesassociated with certain genetic disorders).

Gene expression or replication can be blocked by generating triplexstructures in regions to which required regulatory proteins (ormolecules) are known to bind (for example, HIV transcription associatedfactors like promoter initiation sites and SP1 binding sites, McShan etal., J. Biol. Chem., 267:5712-5721, 1992). Alternatively, specificsequences within protein-coding regions of genes (e.g., oncogenes) canbe targeted as well.

When a phosphoramidate 2′-arabino-fluorooligonucleotide is identifiedthat binds a selected duplex target sequence tests, for example, by thegel band shift mobility assay described above, the analog is furthertested for its ability to form stable triplex structures in vitro. Cellculture and in vivo assay systems, such as those described in U.S. Pat.No. 5,631,135 are used.

Target sites can be chosen in the control region of the genes, e.g., inthe transcription initiation site or binding regions of regulatoryproteins (Helene and Toulme, 1990; Birg et al., 1990; Postel et al.,1991; Cooney et al., 1988). Also, target sites can be chosen such thatthe target also exists in mRNA sequences (i.e., a transcribed sequence),allowing oligonucleotides directed against the site to function asantisense mediators as well (see above).

Also, phosphoramidate 2′-arabino-fluorooligonucleotide molecules can beused to generate triplex molecules with a third strand target (i.e., asingle-strand nucleic acid). For example, a DNA molecule having tworegions capable of forming a triplex structure with a selected targetthird strand molecule can be synthesized. Typically the two regions arelinked by a flexible region which allows the association of the tworegions with the third strand to form a triplex.

Hinge regions can comprise any flexible linkage that keeps the twotriplex forming regions together and allows them to associate with thethird strand to form the triplex. Third strand targets are selected tohave appropriate purine/pyrimidine content so as to allow formation oftriplex molecules.

The flexible linkage may connect the two triplex forming regions(typically, complementary DNA strands) in any selected orientationdepending on the nature of the base sequence of the target. For example,the two triplex forming regions each have 5′ and 3′ ends, these ends canbe connected by the flexible hinge region in the following orientations:5′ to 3′, 3′ to 5′, 3′ to 3′, and 5′ to 5′.

Further, duplex DNA molecules containing at least one phosphoramidate2′-arabino-fluoro nucleotide in each strand can be used as decoymolecules for transcription factors or DNA binding proteins (e.g.,c-myb).

Single-stranded DNA can also be used as a target nucleic acid foroligonucleotides of the present invention, using, for example,phosphoramidate 2′-arabino-fluorooligonucleotide-containing hairpinstructures. Two phosphoramidate 2′-arabino-fluorooligonucleotides can beselected for single-strand DNA target-directed binding. Binding of thetwo phosphoramidate 2′-arabino-fluorooligonucleotides to thesingle-strand DNA target results in formation of a triplex.

D. Pharmaceutical Compositions

The present invention includes pharmaceutical compositions useful inantisense and antigene therapies. The compositions comprise an effectiveamount of N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides incombination with a pharmaceutically acceptable carrier. One or moreN3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides (havingdifferent base sequences) may be included in any given formulation. Inaddition, the 2′-arabino-fluorooligonucleotides of the present inventionmay also be used in combination with one or more other oligonucleotidesthat lack 2′-arabino-fluoro analog phosphoramidate nucleosides.

The N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides, whenemployed in therapeutic applications, can be formulated neat or with theaddition of a pharmaceutical carrier. The pharmaceutical carrier may besolid or liquid. The formulation is then administered in atherapeutically effective dose to a subject in need thereof.

Liquid carriers can be used in the preparation of solutions, emulsions,suspensions and pressurized compositions. The N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides are dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, an organicsolvent, a mixture of both, or pharmaceutically accepted oils or fats.The liquid carrier can contain other suitable pharmaceutical additivesincluding, but not limited to, the following: solubilizers, suspendingagents, emulsifiers, buffers, thickening agents, colors, viscosityregulators, preservatives, stabilizers and osmolarity regulators.Suitable examples of liquid carriers for parenteral administration ofN3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides preparationsinclude water (partially containing additives, e.g., cellulosederivatives, preferably sodium carboxymethyl cellulose solution),alcohols (including monohydric alcohols and polyhydric alcohols, e.g.,glycols) and their derivatives, and oils (e.g., fractionated coconut oiland arachis oil).

For parenteral administration of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides the carrier can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile carriers areuseful in sterile liquid form compositions for parenteraladministration.

Sterile liquid pharmaceutical compositions, solutions or suspensions canbe utilized by, for example, intraperitoneal injection, subcutaneousinjection, intravenously, or topically. For example, antisenseoligonucleotides directed against retinal cytomegalovirus infection maybe administered topically by eyedrops. N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides can be also be administeredintravascularly or via a vascular stent impregnated with mycophenolicacid, for example, during balloon catheterization to provide localizedanti-restenosis effects immediately following injury.

The liquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant. Suchpressurized compositions may also be lipid encapsulated for delivery viainhalation. For administration by intranasal or intrabronchialinhalation or insufflation, N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides may be formulated into an aqueous orpartially aqueous solution, which can then be utilized in the form of anaerosol, for example, for treatment of infections of the lungs likePneumocystis carnii.

N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides may beadministered topically as a solution, cream, or lotion, by formulationwith pharmaceutically acceptable vehicles containing the activecompound. For example, for the treatment of genital warts.

The N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides may beadministered in liposome carriers. The use of liposomes to facilitatecellular uptake is described, for example, in U.S. Pat. Nos. 4,897,355(D. Eppstein et al., issued 30 Jan. 1990) and U.S. Pat. No. 4,394,448(F. Szoka et al., issued 19 Jul. 1983). Numerous publications describethe formulation and preparation of liposomes.

The dosage requirements for treatment with N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides vary with the particular compositionsemployed, the route of administration, the severity of the symptomspresented, the form of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides and the particular subject beingtreated.

In general, N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotidesare administered at a concentration that affords effective resultswithout causing any harmful or deleterious side effects (e.g., aneffective amount). Such a concentration can be achieved byadministration of either a single unit dose, or by the administration ofthe dose divided into convenient subunits at suitable intervalsthroughout the day.

E. Diagnostic Applications

The phosphoramidate 2′-arabino-fluorooligonucleotides of the presentinvention are also useful in diagnostic assays for detection of RNA orDNA having a given target sequence. In one general application, thephosphoramidate 2′-arabino-fluorooligonucleotides are labeled (e.g.,isotopically or other detectable reporter group) and used as probes fornucleic acid samples that bound to a solid support (e.g., nylonmembranes).

Alternatively, the phosphoramidate 2′-arabino-fluorooligonucleotides maybe bound to a solid support (for example, magnetic beads) and homologousRNA or DNA molecules in a sample separated from other components of thesample based on their hybridization to the immobilized phosphoramidateanalogs. Binding of phosphoramidate 2′-arabino-fluorooligonucleotides toa solid support can be carried out by conventional methods. Presence ofthe bound RNA or DNA can be detected by standard methods, for example,using a second labeled reporter or polymerase chain reaction (see U.S.Pat. Nos. 4,683,195 and 4,683,202).

Diagnostic assays can be carried out according to standard procedures,with suitable adjustment of the hybridization conditions to allowphosphoramidate 2′-arabino-fluorooligonucleotide hybridization to thetarget region. The ability of phosphoramidate2′-arabino-fluorooligonucleotides to bind at elevated temperature canalso help minimizes competition for binding to a target sequence betweenthe phosphoramidate 2′-arabino-fluorooligonucleotides probe and anycorresponding single-strand phosphodiester oligonucleotide that ispresent in the diagnostic sample.

F. Other Applications

In one aspect, the phosphoramidate 2′-arabino-fluorooligonucleotides canbe used in methods to enhance isolation of RNA or DNA from samples. Forexample, as discussed above, phosphoramidate2′-arabino-fluorooligonucleotides can be fixed to a solid support andused to isolate complementary nucleic acid sequences, for example,purification of a specific mRNA from a polyA fraction (Goldberg et al.,Methods in Enzymology, 68:206, 1979). The phosphoramidate2′-arabino-fluorooligonucleotides are advantageous for such applicationssince they can form more stable interactions with RNA and duplex DNAthan standard phosphodiester oligonucleotides.

A large number of applications in molecular biology can be found forreporter labeled phosphoramidate 2′-arabino-fluorooligonucleotides,particularly for the detection of RNA in samples. Phosphoramidate2′-arabino-fluorooligonucleotides can be labeled with radioactivereporters (3H, ¹⁴C, ³²P, or ³⁵S nucleosides), biotin or fluorescentlabels (Gryaznov et al., Nucleic Acids Research, 20:3403-3409, 1992).Labeled phosphoramidate 2′-arabino-fluorooligonucleotides can be used asefficient probes in, for example, RNA hybridization reactions (Ausubelet al., Current Protocols in Molecular Biology, Hohn Wiley and Sons,Inc., Media, Pa.; Sambrook et al., in Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Vol. 2, 1989).

Also, double-stranded DNA molecules where each strand contains at leastone phosphoramidate 2′-arabino-fluoronucleotide can be used for theisolation of DNA-duplex binding proteins. In this embodiment a duplexcontaining phosphoramidate 2′-arabino-fluorooligonucleotide is typicallyaffixed to a solid support and sample containing a suspected bindingprotein is then passed over the support under buffer conditions thatfacilitate the binding of the protein to its DNA target. The protein istypically eluted from the column by changing buffer conditions.

The triplex forming DNA molecules described above, containingphosphoramidate 2′-arabino-fluorooligonucleotides, can be used asdiagnostic reagents as well, to, for example, detect the presence of anRNA molecule in a sample.

Further, complexes containing oligonucleotides having N3′→P5′phosphoramidate 2′-arabino-fluoro nucleotides can be used to screen foruseful small molecules or binding proteins: for example, N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotide complexes with duplexDNA can be used to screen for small molecules capable of furtherstabilizing the triplex structure. Similar screens are useful withN3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotide complexesformed with single strand DNA and RNA molecules.

G. Variations

Variations on the phosphoramidate 2′-arabino-fluorooligonucleotides usedin the methods of the present invention include modifications tofacilitate uptake of the oligonucleotide by the cell (e.g., the additionof a cholesterol moiety (Letsinger, U.S. Pat. No. 4,958,013); productionof chimeric oligonucleotides using other intersubunit linkages(Goodchild, Bioconjugate Chem., 1:165-187, 1990); modification withintercalating agents (for example, triplex stabilizing intercalatingagents, Wilson et al., Biochemistry, 32:10614-10621, 1993); and the useof ribose instead of deoxyribose subunits. Further modificationsinclude, 5′ and 3′ terminal modifications to the oligonucleotides (e.g.,—OH, —OR, —NHR, NH₂ and cholesterol).

N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides may also bemodified by conjugation to a polypeptide that is taken up by specificcells. Such useful polypeptides include peptide hormones, antigens andantibodies. For example, a polypeptide can be selected that isspecifically taken up by a neoplastic cell, resulting in specificdelivery of N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides tothat cell type. The polypeptide and oligonucleotide can be coupled bymeans known in the art (see, for example, PCT International ApplicationPublication No. PCT/US89/02363, WO8912110, published Dec. 14, 1989,Ramachandr, K. et al.).

The properties of such modified phosphoramidate2′-arabino-fluorooligonucleotides, when applied to the methods of thepresent invention, can be determined by the methods described herein.

Example 1 General Methods

³¹P NMR spectra were obtained on a Varian 400 MHz spectrometer. ³¹P NMRspectra were referenced against 85% aqueous phosphoric acid. Anionexchange HPLC was performed using a Dionex DX 500 Chromatography System,with a Pharmacia Biotech Mono Q HR 5/5 or 10/16 ion exchange columns.Ion exchange HPLC was performed on the Dionex DX 500 system, using aPharmacia Mono Q HR 5/5 column. Buffer A and B were: 10 mM NaOH, pH12and 10 mM NaOH, 1.5 M NaCl, pH12, respectively. Oligonucleotides wereeluted using a 1.5%/min. linear gradient of buffer B in A; flow 0.5mL/min. Mass spectral analysis was performed by Mass Consortium, SanDiego, Calif. MALDI-TOF analysis of oligonucleotides was obtained usinga PerSpective Biosystems Voyager Elite mass spectrometer, with delayedextraction. Thermal dissociation experiments were conducted on a CaryBio 100 UV-Vis spectrometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic anhydride, 1,2-dichloroethane, and dioxane werepurchased from Aldrich (Milwaukee, Wis.).

All non-2′-arabino-fluorooligonucleotides were synthesized on an ABI 392or 394 DNA synthesizer using standard protocols for the phosphoramiditebased coupling approach (Caruthers, Acc. Chem. Res., 24:278-284, 1991).The chain assembly cycle for the synthesis of oligonucleotidephosphoramidates was the following: (i) detritylation, 3%trichloroacetic acid in dichloromethane, 1 min.; (ii) coupling, 0.1 Mphosphoramidite and 0.45 M tetrazole in acetonitrile, 1-3 min.; (iii)capping, 0.5 M isobutyic anhydride in THF/lutidine, 1/1, v/v, 15 sec;and (iv) oxidation, 0.1 M iodine in THF/pyridine/water, 10/10/1, v/v/v,30 sec.

Chemical steps within the cycle were followed by acetonitrile washingand flushing with dry argon for 0.2-0.4 min. Cleavage from the supportand removal of base and phosphoramidate protecting groups was achievedby treatment with ammonia/EtOH, 3/1, v/v, for 6 h at 55° C. Theoligonucleotides were concentrated to dryness in vacuo after which the2′-t-butyldimethylsilyl groups were removed by treatment with 1M TBAF inTHF for 4-16 h at 25° C. The reaction mixtures were diluted with waterand filtered through a 0.45 nylon acrodisc (from Gelman Sciences, AnnArbor, Mich.). Oligonucleotides were then analyzed and purified by IonExchange HPLC and finally desalted using gel filtration on a PharmaciaNAP-5 or NAP-25 column.

Example 2 Synthesis of 2′-Arabino-Fluoronucleoside PhosphoramiditeMonomers

Preparation of the inventive 2′-arabino-fluoronucleoside monomers isdescribed in FIG. 1. First, a1-α-O-Benzoyl-3,5-O-benzoyl-2-arabinofluoro-2-deoxyfuranose sugarprecursor 1 (FIG. 1) (from Pfanstiehl Laboratories, Inc., Waukegan,Ill.) was converted into a1-α-Bromo-3,5-O-benzoyl-2-arabinofluoro-2-deoxyfuranose intermediate 2with retention of sugar C-1 configuration (Berger et al., Nucl. AcidsRes., 26:2473-2480, 1998). Compound 2 was used, without isolation, in aS_(N)2-type glycosylation reaction with silylated purine and pyrimidinebases according to the literature procedure (Berger et al., Nucl. AcidsRes., 26:2473-2480, 1998), which resulted in formation of2′-arabinofluoro-3′,5′-O-benzoyl nucleosides 3 (FIG. 1). Stereoselectivity of this glycosylation reaction was quite high—more than 90%of the formed nucleoside 3 had the desired α-anomeric configuration, aswas judged by ¹H NMR analysis of crystallized products. Then, 5′- and3′-O-benzoyl protecting groups of nucleoside 3 were removed, almostquantitatively, by methanolic ammonia, and the resultant 5′-,3′-hydroxyl groups containing nucleoside product without additionalpurification was converted into2,3′-anhydro-2′-arabinofluoro-5′-O-benzoyl nucleosides 4 under Mitsunobureaction conditions (Czernecki et al., Synthesis, 239-240, 1991). Thefollowing treatment of the 2,3′-anhydronucleosides with lithium azideresulted in key 2′-arabinofluoro-3′-azido-5′-O-benzoyl precursor 5(Glinski, et al., J. Chem. Soc. Chem. Comm., 915-916, 1970). Thesecompounds were converted into thymidine and uracil2′-arabinofluoro-3′-NH-MMT-5′-O-(cyanoethyl-N,N′-diisopropylamino)-phosphoramidites7 as described in the literature sequence of chemical transformations:catalytic reduction of 3′-azido to 3′-amino group by hydrogen overpalladium, followed by 3′-tritylation, 5′-O-deprotection and5′-O-phosphitylation (Schultz et al., Nucl. Acids Res., 24:2966-2973,1996). Cytosine phosphoramidite 7c was obtained from the 3′-azidoprecursor uracil-to-cytosine conversion process analogous to theliterature procedure for 3′-azido-2′-ribo-fluoronucleosides (Schultz etal., Nucl. Acids Res., 24:2966-2973, 1996) (FIG. 1). Total yields of thecytosine, thymidine and uracil phosphoramidites 7c,u,t were in the rangeof 15-20% as calculated based on the starting sugar precursor 1.Structure of the monomers was confirmed by ¹H, ³¹P, ¹⁹F NMR and by massspectrometric analysis. For example, for thymidine 7, Rp-, Sp-isomers,³¹P NMR, L, PPM (CDCl₃) 149.4; 149.8 ¹⁹F NMR L, ppm (CDCl₃) two hextetsat −190.26 and −190.88). The data for uracil 7 and cystosine 7 is verysimilar: ³¹P NMR, L, PPM (CDCl₃) in range of 148-149; ¹⁹F NMR L, ppm(CDCl₃) two hextets at about −188 and −191.

2′-Arabino-fluoro-amino purine monomers can also be prepared startingwith purine-3′-azido-2′-hydroxyl precursor, orpurine-3′—NH-trityl-2′-hydroxyl precursors that are prepared using thefollowing synthesis methods. The first step of the synthesis involvedtin(IV) chloride or trimethylsilyl triflate mediated glycosylation oftrimethylsilylated nucleobases (Azhayev, et al. (1979) Nucleic AcidsRes., 2:2625-2643; Vorbruggen, et al. (1981) Chem. Ber., 114:1234-1255)to a commonly employed sugar precursor3-azido-1,2-di-O-acetyl-5-O-toluoyl-3-deoxy-D-ribofuranose 10, which wasprepared according to literature procedure (Ozols, et al. Synthesis,557-558). Adenine was protected at N⁶ with a benzoyl group, whileguanine was blocked at N² with an isobutyl group and at O⁶ withdiphenylcarbamate (Zou, et al. (1987) Can. J. Chem., 65:1436-1437). Theprotection of O⁶ with this bulky group allows for selectiveglycosylation to occur at N⁹ with very little (≦10%) formation of theundesired N⁷ regioisomer as judged by TLC analysis. 2,6-Diaminopurinewas protected at each exocylic amine with a phenoxyacetyl group for allglycosylation reactions with this highly polar purine base analogue(Schulhof, et al. (1987) Tetrahedron Lett., 28:51-54).

Two key synthetic improvements were made to the preparation of themonomers, which allowed for rapid access to the final products withimproved overall yields. First, experimental conditions were found whichenabled selective removal of the 2′-O-acetyl protecting group in thepresence of the 5′-O-toluoyl counterpart (Neilson, et al. (1971) Can. J.Chem., 49:493-498) (FIG. 2). This allowed for the omission of a5′-hydroxyl reprotection step from the synthetic protocol. Also, a lowyielding series of steps late in the monomer synthesis, used in theliterature procedure (Gryaznov, et al. (1998) Nucleic Acids Res.,26:4160-4167) to convert a 5′-trityl-nucleoside precursor to the3′-N-trityl-protected amino intermediate, was also averted. Secondly,following the glycosylation reaction, the next five chemicaltransformations were conducted with very high yields. This eliminatedthe need for intermediate purification after steps iv.-viii. (FIG. 2),thus providing a rapid and convenient access to compounds 17a-d.However, it should be noted that for the guanosine and 2,6-diaminopurineanalogues, selective removal of the 2′-O-acetyl protecting group wasunsuccessful. Thus, both 2′-O and 5′-O-protecting groups were removed,after which the 5′-hydroxyl group was selectively reprotected (FIG. 2).

For compound 11a the 2′-O-acetyl group was selectively removed using 50%(v/v) aqueous ammonia in methanol followed by the 3′-azido groupreduction with hydrogen over palladium on carbon. Each of thesereactions proceeded with very high, near quantitative, yields as judgedby TLC and ¹H NMR analysis of the products. The 3′-amino group is thenprotected by treatment with 4-monomethoxytrityl chloride to givecompound 15a. The obtained protected nucleoside precursor was thentreated with diethylamino sulfur trifluoride (DAST) to make anarabino-fluoro adenine nucleoside 16a. The protected arabino-fluoroproduct is concentrated in vacuo to which is added 1.0 M NaOH in 65/30/5pyridine/MeOH/H₂O (70 mL) at 0° C. to remove the 5′-O-toluoyl group. Themixture is stirred for 8 min and quenched by addition of saturatedNH₄Cl. This solution is extracted with ethyl acetate (2×75 mL) and thecombined organic layers are dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue is purified by silica gelchromatography eluting with EtOAc:hexanes (50:50, v/v) to yield2′-arabino-fluoronucleoside 17a. In an alternative method, the fluoroaddition is performed prior to reduction of the 2′ azido group to anamino group (e.g. prior to step v. in FIG. 2).

The inability to selectively remove the 2′-O-acetyl group fromintermediates 11g and 11d, necessitated the following syntheticprotocol. Both 2′-O— and 5′-O-protecting groups were removed with 1 Msodium hydroxide, after which a 5′-O-anisoyl group was selectivelyreintroduced under Mitsunobu conditions to give 13g and 13d. Compounds11g and 11d (about 2.8 g, 3.81 mmol) are dissolved in a 1.0 M NaOHsolution (65/30/5 pyridine/MeOH/H₂O, v/v/v, (40 mL)) at 0° C. Themixtures are stirred for 10 min, and then quenched by addition ofsaturated NH₄Cl (400 mL). The solutions are extracted with CH₂Cl₂ (5×100mL) and the combined organic layers are dried over Na₂SO₄, filtered, andconcentrated in vacuo. For guanine nucleosides the residues istriturated well with Et₂OH (50 mL) to remove diphenylamine and theresulting material dissolved in acetonitrile (25 mL) andtriphenylphosphine (1.2 g, 4.65 mmol). For diaminopurine nucleosides theNa₂SO₄ dried, filtered and concentrated material is dissolvedindimethylformamide (50 ml) and triphenylphosphine (1.5 g, 5.7 mmol) areadded. For the remainder of the steps leading to 13g,d the steps are thesame. p-Anisic acid (0.71 g, 4.65 mmol) and diisopropyl azodicarboxylate(0.92 mL, 4.65 mmol) are dissolved in acetonitrile (5 mL) and addeddropwise to the reaction mixtures. The solutions are stirred at roomtemperature for 1 h and was then quenched by pouring them into saturatedNaHCO₃ (200 mL). The mixtures are extracted with ethyl acetate (200 mL)and after separation the organic phases are washed with saturated NaCl(150 mL). The organic phases are then dried over Na₂SO₄ and concentratedin vacuo. The residues are purified by silica gel chromatography elutingwith a gradient of EtOAc:Hexanes:MeOH (49:49:2, v/v/v to 47.5:47.5:5,v/v/v) to afford 13g,d.

It should be noted that the high reactivity of the 2′-hydroxyl group ofthe 3′-azido-2′-hydroxyl guanosine intermediate prevented selectivereprotection of the 5′-hydroxyl group by either benzoyl chloride orbenzoyl anhydride. The same series of steps described above for adinineare used to convert 13 g and 13d into 17g and 17d respectively (FIG. 2).The MMT protecting group in 17a-d can be removed to give the unprotectedamino sugar moiety. The 5′-OH group can then be selectively protectedwith an alkyl group or converted to a mono, di, or triphosphate group.The final step for monomer preparation involves phosphitylation of 17a-dto give the 5′-(2-cyanoethyl-N,N′-diisopropylamino)nucleosidephosphoramidite building blocks 18a-d (FIG. 2).

In order to gain information on the2′-arabino-fluoro-3′-aminonucleosides sugar puckering, a model N3′→P5′phosphoramidate tri-nucleotide dTn-a(U^(f)nU^(f)n) [SEQ ID NO:1],containing internucleoside and terminal2′-arabino-fluoro-3′-aminonucleosides (³¹P NMR, L, ppm in D₂O 7.8 and6.7) was synthesized, and the compound was analyzed by high resolution¹H and ³¹P NMR spectroscopy. The analysis revealed vicinal protoncoupling constants J³H1′-H2″ of 4.68 Hz for both2′-arabino-fluoro-uridine nucleosides, and J³H2″-H3′ 3.81 Hz and 4.46 Hzfor internucleoside and for 3′-terminal 2′-arabino-fluoro-uridines,respectively. The corresponding coupling constants for the preparedmodel di-nucleoside aU^(f)pdT [SEQ ID NO:2], containing2′-arabino-fluoro-3′-hydroxy uracil and the phosphodiesterinternucleoside bond, were 3.67 Hz and 1.83 Hz respectively, whichindicates a significant difference in 3′—NH— and3′-O-2′-ara-fluoronucleosides sugar puckering. The observed couplingconstants for the tri-nucleotide alone do not define the2′-arabino-fluoro-3′-amino sugars conformation. However, the dataobtained from 2D COSY spectra of the tri-nucleotide large intensity ofH3′-H4′ cross peaks, as well as the data from ¹H-³¹P 2D heteronuclearspectra (strong H3′-(i-1)P and weak H4′-P cross peaks) suggestprevalence of N-type conformation for the2′-arabino-fluoro-3′-aminonucleosides. The substitution of 3′-oxygen by3′-amino group apparently shifts the 2′-arabino-fluoro furanoseconformational equilibrium towards N-type, or C3′-endo, for2′-arabino-fluoronucleosides, which is similar to that for the2′-deoxy-3′-aminonucleosides.

Example 3 Synthesis of N3′→P5′ Phosphoramidite2′-Arabino-Fluorooligonucleotides

The oligo-2′-arabino-fluoro nucleotide N3′→P5′ phosphoramidates wereassembled similarly to the oligo-2′-ribo-fluoro N3′→P5′ phosphoramidatecompounds. Solid phase synthesis was based on the phosphoramiditetransfer reaction using monomer building blocks composed of5′-(O-cyanoethyl-N,N′-diisopropylamino)-phosphoramidites of3′-MMTr-protected-3′-amino-2′-arabino-fluoronucleosides (Schultz et al.,Nucl. Acids Res., 24:2966-2973, 1996).

The oligonucleotide synthesis was conducted on an automated DNA/RNA ABI394 synthesizer using the synthetic cycle described before for the2′-ribo-fluoro phosphoramidites with step-wise coupling yields of about95-97%. Each of the prepared 2′-arabino-fluorooligonucleotides weresynthesized starting from the 5′-end using a support-bound2′-deoxy-3′-aminonucleoside as the 5′-terminal residue. Coupling stepsinvolved exchange of the diisopropylamino group of the approaching5′-O-phosphoramidite monomer for the 3′-amino group of the support boundnucleoside. Standard RNA synthesis coupling times (10 min) and activator(1H-tetrazole) were used for each synthetic cycle. Unreacted 3′-aminogroups were then capped with isobutyric anhydride, after which oxidationof the internucleotide phosphoramidite diester linkage into thephosphoramidate group was carried out with aqueous iodine. Subsequentdetritylation of the 3′-amino group of the added residue enabledadditional chain elongation steps to be repeated for the construction ofthe desired 2′-arabino-fluororooligonucleotide phosphoramidates.

Compounds were cleaved from a solid phase support and all the protectivegroups were removed with concentrated aqueous ammonia, about 0.5 to 4.5hours at 55° C. The resulting 2′-arabino-fluorooligonucleotide N3′→P5′phosphoramidates have the structure shown in FIG. 3. The inventiveoligonucleotide structure shown in FIG. 3 was determined by firstpurifying and then analyzing the cleaved and deprotected reactionmixtures. The synthesized oligos were purified by ion exchange (IE)HPLC. The structure of the IE HPLC isolatedoligo-2′-arabino-fluoronucleotide N3′→P5′ phosphoramidates was confirmedby ³¹P and ¹⁹F NMR analysis.

The sequences of representative inventive oligonucleotides that havebeen prepared are summarized in Table 1.

TABLE 1 OLIGONUCLEOTIDES AND MELTING TEMPERATURE (Tm) VALUES OF THEIRCOMPLEXES T_(m), ° C.^(b) T_(m), ° C.^(b) Expt.5′-Oligonucleotide-3′^(a) RNA DNA dsDNA^(e) 1 d(UpUpUpUpUpUpUpUpUpT)17.9;^(c) 20.3^(d) 16.7;^(c) 24.6^(d) n.o;^(c) n.o^(d) [SEQ ID NO: 3] 2r(Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)T) 32.9; 36.6n.o; 18.0 n.o; n.o [SEQ ID NO: 4] 3a(Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)T) 27.5; 28.617.0; 25.0 <14; 20.9 [SEQ ID NO: 5] 4 d(UnUnUnUnUnUnUnUnUnT) 38.1; 47.218.5; 38.2 <15; n.d [SEQ ID NO: 6] 5dT-r(Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)) 55.5; 61.937.4; 56.3 42.2; 54.1 [SEQ ID NO: 7] 6dT-a(Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)Un^(f)) 40.0; 40.025.7; 37.0 22.0; 31.2 [SEQ ID NO: 8] 7 d(CnUnCnUnCnUnGnCnCn) 66.7 n.d —[SEQ ID NO: 9] 8 a(Cn^(f)Un^(f)Cn^(f)Un^(f)Cn^(f)Un^(f)GnCn^(f)Cn^(f))66.8 n.d — [SEQ ID NO: 10] ^(a)d, r, and a correspond to the 2′-deoxy,2′-ribo-fluoro and to 2′-arabino-fluorornucleosides respectively; p andn correspond to the internucleoside phosphodiester and N3′→P5′phosphoramidate linkages, respectively; ^(b)melting temperature, T_(m)(±0.5° C.), of the duplexes formed with natural phosphodiester ssDNA orRNA strands, or: ^(e)triplexes formed with dA/dT duplex part ofd(A₁₀C₄T₁₀) hairpin. Buffers: ^(c)150 mM NaCl, 10 mM sodium phosphatebuffer pH 7.4 - first number, or in: ^(d)the same buffer containing anadditional 10 mM magnesium chloride - second number; n.o or n.d - themelting transitions and T_(m)'s were not observed or not determined,respectively.

First, the compounds were characterized by ion exchange (IE) HPLCanalysis. Oligo-2′-arabino-fluoronucleotide N3′→P5′ phosphoramidate [SEQID NO:6], Table 1, has significantly longer, by about 8 minutes,retention time on IE HPLC column at pH 12, than that for theisosequential 2′-ribo-fluoro phosphoramidate counterpart oligonucleotide[SEQ ID NO:7], Table 1. At the same time these oligonucleotidespractically co-elute from the same column at pH 7 buffer conditions. IEHPLC was carried out on Dionex DX 500 system, using Pharmacia MonoQ 5/5column; Buffer A: 10 mM NaOH, pH12; Buffer B: 10 mM NaOH, 1.5 M NaCl,pH12; 1.5%/min. linear gradient of buffer B in A; flow 0.5 mL/min.Retention time (Rt), for co-injected oligonucleotides SEQ ID NOs:7, 8, 4and 5, Table 1, was 33.6, 41.5, 42.5 and 46.5 minutes, respectively. AtpH 7.2 (10 nM Na-phosphate buffer) retention times for oligonucleotidesSEQ ID NOs:7 and 8 were 23.7 and 23.8 minutes, respectively. Thedifferent degrees of uracil base ionization under the alkalineconditions, as well as ionization of internucleoside 3′-NHP(O)O-5′groups, which are influenced by the spatial orientation of the2′-fluorine likely determine the differences in the oligomerschromatographic behavior. Probably, the 2′-fluorine atom increasesacidity of uracil bases and in trans-oriented internucleosidephosphoramidate groups better in arabino- than in ribo-configuration.Additionally, retention times (at pH 12) for the 2′-ribo-fluoro and2′-arabino-fluoro phosphodiester decanucleotides SEQ ID NOs:4 and 5,Table 1, were noticeably longer, than for their phosphoramidate cognateoligonucleotides SEQ ID NOs:7 and 8, Table 1. This difference likelyreflects a higher lypophilicity of 2′-fluoro phosphodiesteroligonucleotides. Also, similarly to the phosphoramidates,oligo-2′-arabino-fluoronucleotide was retained longer than the2′-ribo-fluoro isomer on IE HPLC column, indicating its highernet-negative charge in alkaline conditions.

Example 4 Stability and Duplex Formation Properties of N3′→P5′Phosphoramidate 2′-Arabino-Fluorooligonucleotides

It was reported that the 2′-ribo-fluoropyrimidine containingoligonucleotides with N3′→P5′ phosphoramidate or with phosphodiesterinternucleoside linkages are somewhat labile during treatment withaqueous ammonia at 55° C. (Schultz et al., Nucl. Acids Res.,24:2966-2973, 1996; Krug et al., Nucleosides Nucleotides, 8:1473-1483,1989). Under these basic conditions pyrimidine-O2 mediated eliminationof the 2′-fluorine atom takes place, resulting in formation of2,3′-O-anhydronucleosides and multiple products of their consequentreactions with aqueous ammonia, including 2′-arabino-hydroxylnucleosides (Krug et al., Nucleosides Nucleotides, 8:1473-1483, 1989).In contrast, the prepared 2′-arabino-fluoro phosphoramidateoligonucleotides were stable under these conditions. The2′-arabino-fluoro phosphoramidate decanucleotide SEQ ID NO:8, Table 1,was practically intact after exposure to concentrated aqueous ammoniafor 24 hours, 55° C., whereas isomeric 2′-ribo-fluoro counterpart SEQ IDNO:7, Table 1, was converted into a very complex mixture of productsunder these conditions, as was judged by IE HPLC analysis. Moreover, thehalf-life of 2′-arabino-fluoro phosphoramidate SEQ ID NO:8 in acidicmedia, pH 3, was about 2 and 9 times longer (about 280 minutes), thanthat for the isosequential 2′-ribo-fluoro phosphoramidateoligonucleotide SEQ ID NO:7 (about 135 minutes) and for 2′-deoxyphosphoramidate oligonucleotide SEQ ID NO:6 (about 30 minutes),respectively (see Schultz, et al. 1996 for acid stability method).

The ability of N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotidesto form complexes with complementary DNA and RNA strands was examinedand compared with related oligonucleotide analogues using thermaldenaturation experiments. The results of the study are summarized inTable 1. Substitution of 2′-deoxy nucleosides by their 2′-ribo-fluorocognates in phosphodiester oligonucleotide, compounds SEQ ID NOs:3 and4, resulted in significant stabilization of duplexes with RNA—T_(m) ofabout 15.0-16.3° C., but destabilization of complexes with DNA—T_(m) ofabout −6.7-16° C. (compare experiments 1 and 2, Table 1).2′-arabino-fluoro and 2′-deoxy phosphodiester oligonucleotides SEQ IDNOs:5 and 10 formed duplexes of similar stability with DNA, but, withRNA the T_(m) of the 2′-arabino-fluorooligonucleotide complex was about8.3-9.6° C. higher than the 2′-deoxy phosphodiester oligonucleotide(compare experiments 1 and 3, Table 1). Substitution of2′-deoxy-3′-aminonucleosides by 2′-arabino-fluoro-3′-amino counterpartsstabilized duplexes, by about 2-7° C., with DNA and RNA in a low ionicstrength buffer. In presence of an additional 10 mM magnesium chloride a2′-deoxy phosphoramidate duplex with RNA is more stable, by about 7° C.,than the duplex formed by a 2′-arabino-fluorooligonucleotide counterpart(compare experiments 4 and 6, Table 1). Interestingly, increasing thebuffer ionic strength unexpectedly had no effect on T_(m) value ofoligonucleotide SEQ ID NO:6 duplex with RNA. Also, 2′-arabino-fluorophosphoramidate SEQ ID NO:6 duplexes were more stable than those formedby a 2′-arabino-fluoro phosphodiester oligonucleotide SEQ ID NO:5,−T_(m) 8.7-12.5° C. (compare experiments 3 and 6, Table 1). The moststable complexes with both DNA and RNA strands, −T_(m) of about 14-21°C., were formed by 2′-ribo-fluoro phosphoramidate oligonucleotide SEQ IDNO:7 (compare experiments 5, 6 and 7, Table 1). Duplexes formed by themixed base 2′-arabino-fluoro and 2′-deoxy phosphoramidateoligonucleotides SEQ ID NOs:9 and 10 have a similar thermal stability,which likely reflects similarity of the nucleosides sugar puckering, asindicated by the NMR analysis (compare experiments 7 and 8, Table 1).Moreover, 2′-arabino-fluoro phosphoramidate oligonucleotide SEQ ID NO:8formed a more stable triplex with dsDNA than did a 2′-deoxyphosphoramidate oligonucleotide SEQ ID NO:6, or phosphodiesteroligonucleotides SEQ ID NOs:3-5, but not a 2′-ribo-fluoro counterpartoligonucleotide SEQ ID NO:7 (Table 1). These data demonstrate thesynergistic duplex and triplex stabilizing effects of 2′-arabino-fluoroand 3′-amino modifications.

Example 5 Preparation of Affinity Purified Extract Having TelomeraseActivity

Extracts used for screening telomerase inhibitors were routinelyprepared from 293 cells over-expressing the protein catalytic subunit oftelomerase (hTERT). These cells were found to have 2-5 fold moretelomerase activity than parental 293 cells. 200 ml of packed cells(harvested from about 100 liters of culture) were resuspended in anequal volume of hypotonic buffer (10 mM Hepes pH 7.9, 1 mM MgCl₂, 1 mMDTT, 20 mM KCl, 1 mM PMSF) and lysed using a dounce homogenizer. Theglycerol concentration was adjusted to 10% and NaCl was slowly added togive a final concentration of 0.3 M. The lysed cells were stirred for 30min. and then pelleted at 100,000×g for 1 hr. Solid ammonium sulfate wasadded to the S100 supernatant to reach 42% saturation. The material wascentrifuged; the pellet was resuspended in one fifth of the originalvolume and dialyzed against Buffer ‘A’ containing 50 mM NaCl. Afterdialysis the extract was centrifuged for 30 min. at 25,000×g. Prior toaffinity chromatography, Triton X-100 (0.5%), KCl (0.3 M) and tRNA (50μg/ml) were added. Affinity oligo (5′ biotinTEG-biotinTEG-biotinTEG-GTAGAC CTG TTA CCA guu agg guu ag 3′ [SEQ ID NO:11]; lower case represents2′ O-methyl ribonucleotides and upper case represents deoxynucleotides)was added to the extract (1 mmol per 10 ml of extract). After anincubation of 10 min. at 30° C., Neutravidin beads (Pierce; 250 μl of a50% suspension) were added and the mixture was rotated overnight at 4°C. The beads were pelleted and washed three times with Buffer ‘B’containing 0.3 M KCl, twice with Buffer ‘B’ containing 0.6 M KCl, andtwice more with Buffer B containing 0.3 M KCl. Telomerase was eluted inBuffer ‘B’ containing 0.3 M KCl, 0.15%. Triton X-100 and a 2.5 molarexcess of displacement oligo (5′-CTA ACC CTA ACT GGT AAC AGG TCT AC-3′[SEQ ID NO:12] at 0.5 ml per 125 μl of packed Neutravidin beads) for 30min. at room temperature. A second elution was performed and pooled withthe first. Purified extracts typically had specific activities of 10fmol nucleotides incorporated/min./μl extract, or 200nucleotides/min./mg total protein.

Buffer ‘A’ Buffer ‘B’ 20 mM Hepes pH 7.9 20 mM Hepes pH 7.9 1 mM MgCl2 1mM EDTA 1 mM DTT 1 mM DTT 1 mM EGTA 10% glycerol 10% glycerol 0.5%Triton X-100

Example 6

Telomerase Inhibition by Oligonucleotide N3′→P5′ 2′-Arabino-FluoroPhosphoramidates

Three separate 100 μl telomerase assays are set up with the followingbuffer solutions: 50 mM Tris acetate, pH 8.2, 1 mM DTT, 1 mM EGTA, 1 mMMgCl₂, 100 mM K acetate, 500 μM dATP, 500 μM TTP, 10 μM ³²P-dGTP (25Ci/mmol), and 100 nM d(TTAGGG)₃ [SEQ ID NO: 13]. To the individualreactions 2.5, 5 or 10 μl of affinity-purified telomerase (see Example5) is added and the reactions are incubated at 37 C. At 45 and 90minutes, 40 μl aliquots are removed from each reaction and added to 160μl of Stop Buffer (100 mM NaCl, 10 mM Na pyrophosphate, 0.2% SDS, 2 mMEDTA, 100 μg/ml tRNA). 10 μl trichloroacetic acid (TCA) (100%) is addedand the sample is incubated on ice for 30 minutes. The sample ispelleted in a microcentrifuge (12000×g force) for 15 minutes. The pelletis washed with 1 ml 95% ethanol and pelleted again in themicrocentrifuge (12000×g force) for 5 minutes. The pellet is resuspendedin 50 μl dH₂O and transferred to a 12×75 glass test tube containing 2.5ml of ice cold solution of 5% TCA and 10 mM Na pyrophosphate. The sampleis incubated on ice for 30 minutes. The sample is filtered through a 2.5cm wet (dH₂O) GFC membrane (S&S) on a vacuum filtration manifold. Thefilter is washed three times under vacuum with 5 ml ice cold 1% TCA, andonce with 5 ml 95% ethanol. The filter is dried and counted in ascintillation counter using scintillation fluid. The fmol of nucleotideincorporated is determined from the specific activity of radioactivetracer. The activity of extract is calculated based on the dNTPincorporated and is expressed as fmol dNTP/min./μl extract.

Telomerase Activity Assay

Bio-Tel FlashPlate Assay

An assay is provided for the detection and/or measurement of telomeraseactivity by measuring the addition of TTAGGG telomeric repeats to abiotinylated telomerase substrate primer; a reaction catalyzed bytelomerase. The biotinylated products are captured instreptavidin-coated microtiter plates. An oligonucleotide probecomplementary to 3.5 telomere repeats labeled with [³³P] is used formeasuring telomerase products, as described below. Unbound probe isremoved by washing and the amount of probe annealing to the capturedtelomerase products is determined by scintillation counting.

Method:

-   I. 2′-Arabino-fluoro phosphoramidate oligonucleotides were stored as    concentrated stocks and dissolved in PBS.-   II. For testing, the 2′-arabino-fluoro phosphoramidate    oligonucleotides were diluted to a 15× working stock in PBS and 2 μl    was dispensed into two wells of a 96-well microtiter dish (assayed    in duplicate).-   III. Telomerase extract was diluted to a specific activity of    0.04-0.09 fmol dNTP incorporated/min./μl in Telomerase Dilution    Buffer and 18 PI added to each sample well to preincubate with    compound for 30 minutes at room temperature.-   IV. The telomerase reaction was initiated by addition of 10 μl    Master Mix to the wells containing telomerase extract and    oligonucleotide compound being tested. The plates were sealed and    incubated at 37° C. for 90 min.-   V. The reaction was stopped by the addition of 10 μl HCS.-   VI. 25 μl of the reaction mixture was transferred to a 96-well    streptavidin-coated FlashPlate (NEN) and incubated for 2 hours at    room temperature with mild agitation.-   VII. The wells were washed three times with 180 μl 2×SSC without any    incubation.-   VIII. The amount of probe annealed to biotinylated telomerase    products were detected in a scintillation counter.-   IX.    Buffers:

Telomerase Dilution Buffer

50 mM Tris-acetate, pH 8.2

1 mM DTT

1 mM EGTA

1 mM MgCl₂

830 nM BSA

Master Mix (MM)

50 mM Tris-acetate, pH 8.2

1 mM DTT

1 mM EGTA

1 mM MgCl₂

150 mM K acetate

10 μM dATP

20 μM dGTP

120 μM dTTP

100 nM biotinylated primer (5′-biotin-AATCCGTCGAGCAGAGTT-3′) [SEQ IDNO:14]

5.4 nM labeled probe [5′-CCCTAACCCTAACCCTAACCC-(³³P) A₁₋₅₀-3′][SEQ IDNO:15]; specific activity approximately 10⁹ cpm/μg or higher

Hybridization Capture Solution (HCS)

12×SSC (1×=150 mM NaCl/30 mM Na₃Citrate)

40 mM EDTA

40 mM Tris-HCl, pH 7.0

Using the foregoing assay, IC₅₀ values for the inventive2′-arabino-fluoro phosphoramidate oligonucleotides represented by thesequences shown in Table 2 can be determined.

TABLE 2 2′-ARABINO-FLUOROOLIGONUCLEOTIDES 1-4 ASTELOMERASE INHIBITORS IN COMPARISON WITH2′-RIBOSE-FLUORO PHOSPHORAMIDATES: SEQ ID NOs 5′-Oligonucleotide-3′^(a)SEQ ID NO: 16dG-a(Tn^(f)Tn^(f)An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f))SEQ ID NO: 17dG-a(Tn^(f)Tn^(f)Gn^(f)An^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)Gn^(f))SEQ ID NO:18dT-a(An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f)An^(f)Cn^(f)An^(f)An^(f)) SEQ ID NO: 19dT-a(An^(f)Gn^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)An^(f)Gn^(f)Cn^(f)An^(f)An^(f)) SEQ ID NO: 20dG-r(Tn^(f)Tn^(f)An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f))SEQ ID NO: 21dG-r(Tn^(f)Tn^(f)Gn^(f)An^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)Gn^(f))SEQ ID NO: 22dT-r(An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f)An^(f)Cn^(f)An^(f)An^(f)) SEQ ID NO: 23dT-r(An^(f)Gn^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)An^(f)Gn^(f)Cn^(f)An^(f)An^(f)) ^(a)d, r, and a correspond to the 2′deoxy, 2′ribo-fluoroand to 2′-arabino-fluorornucleosides respecitvely; p and n correspond tothe internucleoside phosphodiester and N3′→P5′phosphoramidate linkages,respecitvely.

Oligonucleotides SEQ ID NOs:17 and 19 are mismatch controls foroligonucleotides SEQ ID NOs:16 and 18, respectively, that are used tocompare the telomerase inhibiting power of 2′-arabino-fluorophosphoramidate oligonucleotides to corresponding 2′-ribose-fluorophoshoramidate analog oligonucleotides. Similarly, oligonucleotides SEQID NOs:21 and 23 are mismatch controls for the oligonucleotides SEQ IDNOs:20 and 22.

Based upon the thermal stability of the inventive oligonucleotides(Table 1), it is anticipated that a telomerase inhibition assayperformed using 2′-arabino-fluoro phosphoramidate polynucleotides SEQ IDNOs:16 and 18 of Table 2 will be more effective at inhibiting telomeraseactivity than their counterpart 2′-ribose-fluorooligonucleotides SEQ IDNO:20 and 22. Thus, the 2′-arabino-fluoro phosphoramidateoligonucleotides of the present invention are expected to be not onlymore active in the telomerase inhibition assay as compared to their2′-ribose-fluorooligonucleotides counterparts, but are also more acidresistant than them as well (see Table 1). This combination ofcharacteristics imparts the inventive 2′-arabino-fluoro phosphoramidateoligonucleotides with an important advantage compared to2′-ribose-fluoro phosphoramidate polynucleotides.

Example 7 Anti-Tumor Activity of 2′-Arabino-Fluoro PhosphoramidateOligonucleotides

Ex Vivo Studies

a. Reduction of Telomere Length in Tumor Cells

Colonies of the tumor cell lines, such as the ovarian tumor cell linesOVCAR-5, and SK-OV-3, and normal human cells used as a control (e.g.,normal human BJ cells) are prepared using standard methods andmaterials. In one test, the colonies are prepared by seeding15-centimeter dishes with about 10⁶ cells in each dish. The dishes areincubated to allow the cell colonies to grow to about 80% confluence, atwhich time each of the colonies are divided into two groups. One groupis exposed to a subacute dose of a 2′-arabino-fluoro phosphoramidateoligonucleotide of the invention at a predetermined concentration (e.g.,between about 100 nM and about 20 μM) for a period of about 4-8 hoursafter plating following the split; the other group is exposed to acontrol (e.g., a phosphoramidate oligonucleotide complementary totelomerase RNA but having at least one base sequence that is mismatchedrelative to the sequence of telomerase RNA.

Each group is then allowed to continue to divide, and the groups aresplit evenly again (near confluence). The same number of cells areseeded for continued growth. The test 2′-arabino-fluoro phosphoramidateoligonucleotide or control oligonucleotide is added every fourth day tothe samples at the same concentration delivered initially. Remainingcells are analyzed for telomere length. As the untested cell culturesnear confluence, the samples are split again as just described. Thissequence of cell doubling and splitting is continued for about 20 to 25doublings. Thus, a determination of telomere length as a function ofcell doublings is obtained.

Telomere length is determined by digesting the DNA of the cells usingrestriction enzymes specific for sequences other than the repetitive T₂AG₃ sequence of human telomeres (TRF analysis). The digested DNA isseparated by size using standard techniques of gel electrophoresis todetermine the lengths of the telomeric repeats, which appear, afterprobing with a telomere DNA probe, on the gel as a smear ofhigh-molecular weight DNA (approximately 2 Kb-15 Kb).

The results of the telomere length analysis are expected to indicatethat the 2′-arabino-fluoro phosphoramidate oligonucleotides of theinvention have no affect on the rate of decrease in telomere length forcontrol cells as a function of progressive cell doublings. With respectto the tumor cell lines, however, measurable decreases in telomerelength are expected to be determined for tumor cells exposed to the2′-arabino-fluoro phosphoramidate oligonucleotides of the invention.Tumor cells exposed to the control oligonucleotides are expected tomaintain steady telomere lengths. Thus, the compounds of the inventionare expected to cause resumption of the normal loss of telomere lengthas a function of cell division in tumor cells.

In another experiment, HEK-293 cells are incubated with a2′-arabino-fluoro phosphoramidate oligonucleotide of the invention and acontrol oligonucleotide at concentrations between about 0.1 μM and about20 μM using the protocol just described. Cells are expected to entercrisis (i.e., the cessation of cell function) within several weeksfollowing administration of the test 2′-arabino-fluoro phosphoramidateoligonucleotides of the invention. In addition, TRF analysis of thecells using standard methodology is expected to show that the test2′-arabino-fluoro phosphoramidate oligonucleotides of the invention areeffective in reducing telomere length. In addition to the HEK-293 cellsdescribed above, this assay can be performed with anytelomerase-positive cell line, such as HeLa cells.

b. Specificity

Phosphoramidate 2′-arabino-fluoro oligonucleotides of the invention arescreened for activity (IC₅₀) against telomerase and other enzymes knownto have RNA components by performing hybridization tests or enzymeinhibition assays using standard techniques. Oligonucleotides havinglower IC₅₀ values for telomerase as compared to the IC₅₀ values towardthe other enzymes being screened are said to possess specificity fortelomerase.

c. Cytotoxicity

The XTT assay for cytotoxicity is performed using HeLa cells. The celllines used in the assay are exposed to a 2′-arabino-fluorophosphoramidate oligonucleotide of the invention for 72 hours atconcentrations ranging from about 1 μM to about 100 μM. During thisperiod, the optical density (OD) of the samples is determined for lightat 540 nanometers (nm). No significant cytotoxic effects are expected tobe observed at concentrations less than about 20 μM. It will beappreciated that other tumor cells lines such as the ovarian tumor celllines OVCAR-5 and SK-OV-3 can be used to determine cytotoxicity inaddition to control cell lines such as normal human BJ cells. Otherassays for cytotoxicity such as the MTT assay (see Berridge et al.,Biochemica 4:14-19, 1996) and the alamarBlue™ assay (U.S. Pat. No.5,501,959) can be used as well.

Preferably, to observe any telomerase inhibiting effects the2′-arabino-fluoro phosphoramidate oligonucleotides should beadministered at a concentration below the level of cytotoxicity.Nevertheless, since the effectiveness of many cancer chemotherapeuticsderives from their cytotoxic effects, it is within the scope of thepresent invention that the phosphoramidate oligonucleotides of thepresent invention be administered at any dose for which chemotherapeuticeffects are observed.

In Vivo Studies

A human tumor xenograft model in which OVCAR-5 tumor cells are graftedinto nude mice can be constructed using standard techniques andmaterials. The mice are divided into two groups. One group is treatedintraperitoneally with a 2′-arabino-fluoro phosphoramidateoligonucleotides of the invention. The other group is treated with acontrol comprising a mixture of phosphate buffer solution (PBS) and anoligonucleotide complementary with telomerase RNA but has at least a onebase mismatch with the sequence of telomerase RNA. The average tumormass for mice in each group is determined periodically following thexenograft using standard methods and materials.

In the group treated with a 2′-arabino-fluoro phosphoramidateoligonucleotide of the invention, the average tumor mass is expected toincrease following the initial treatment for a period of time, afterwhich time the tumor mass is expected to stabilize and then begin todecline. Tumor masses in the control group are expected to increasethroughout the study. Thus, the 2′-arabino-fluoro phosphoramidateoligonucleotides of the invention are expected to lessen dramaticallythe rate of tumor growth and ultimately induce reduction in tumor sizeand elimination of the tumor.

Thus, the present invention provides novel 2′-arabino-fluorophosphoramidate oligonucleotides and methods for inhibiting telomeraseactivity and treating disease states in which telomerase activity hasdeleterious effects, especially cancer. The phosphoramidate2′-arabino-fluoro oligonucleotides of the invention provide a highlyselective and effective treatment for malignant cells that requiretelomerase activity to remain immortal; yet, without affectingnon-malignant cells.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A compound of the formula:

wherein: B is a purine or pyrimidine or a base analog thereof; R₂ islower alkyl, PO₃, or PN(R₄)₂OR₅ wherein R₄ is alkyl, and R₅ iscyano-lower alkyl; and R₃ is hydrogen or substituted or unsubstitutedtrityl.
 2. A compound according to claim 1, wherein R₂ is PN(R₄)₂OR₅wherein R₄ is isopropyl, R₅ is β-cyanoethyl and R₃ is monomethoxytrityl.3. A compound according to claim 1, wherein the constituent B isexocyclic amino protected.
 4. A compound according to claim 1, wherein Bis N₂-isobutyrylguanine, B is 2,6-diaminopurine and the exocyclic aminegroups of 2,6-diaminopurine are protected with a phenoxyacetyl group, orB is cytosine and the N4 amino group of cytosine is protected with abenzoyl group.
 5. A compound of the formula:

wherein: B is a purine or pyrimidine or a base analog thereof; R₂ isPN(R₄)₂OR₅ wherein R₄ is isopropyl, and R₅ is β-cyanoethyl; and R₃ ishydrogen or substituted or unsubstituted trityl.